It is known that nitric oxide is formed enzymatically in vivo from arginine as a normal metabolite which is an important component of endothelium-derived relaxing factors (EDRFs). The EDRFs are believed to participate in regulation of blood flow and vascular resistance. In addition to vascular endothelium, macrophages have also been shown to produce nitric oxide in the body which is a component of their cell killing and/or cytostatic function.
The enzyme which forms nitric oxide from arginine, i.e., nitric oxide synthase, is believed to occur in two distinct isoforms, namely the constitutive isoform and the inducible isoform.
The constitutive isoform is present in normal endothelial cells, neurons and some other tissues. The formation of nitric oxide by the constitutive isoform in endothelial cells is thought to play a role in normal blood pressure regulation.
The inducible isoform of nitric oxide synthase has been isolated from activated macrophages and is induced by various cytokines or combinations of cytokines in endothelial cells and vascular smooth muscle cells. It is believed that in septic shock or cytokine-induced shock that the observed life-threatening hypotension is due mainly or wholly to production of larger than normal amounts of nitric oxide by the inducible isoform of nitric oxide synthase.
Septic shock is an acute and serious cardiovascular collapse resulting from the systemic response to a bacterial infection, and is manifested by hypotension, a reduced response to vasoconstrictors, generalized tissue damage and multi-organ failure. It is the most common cause of death in the intensive-care unit; 400,000 cases of septicemia per year in the United States with mortality rates between 25% and 50%. The steadily increasing incidence of septic shock stems from an increasing proportion of elderly in the population, increasing frequency of invasive surgical procedures, extensive use of immunosuppressive and chemotherapeutic agents, and increasing prevalence of chronic debilitating conditions. Because the mechanisms underlying sepsis and septic shock are not yet known, therapeutic interventions have been largely ineffective.
A common cause of septic shock is Gram-negative bacterial infection. The lipopolysaccharide (LPS), an integral part of the outer layer of the Gram-negative bacterial cell wall, is instrumental in bringing about septic shock. Upon lysis of the bacteria, LPS is released into the surrounding medium, forming a micelle-like solute, known as endotoxin. Intravenous administration of LPS in animals and man produces a shock-like syndrome. Despite extensive studies, the reasons for the vascular effects of endotoxaemia are not clear, which have been attributed to increasing circulating levels of vasoactive angiotensin II and catecholamines, bradykinin, platelet activating factor, prostaglandins and leukotrienes, thromboxane A2, endothelin, phospholipase A.sub.2, and more recently, to the cytokines such as interleukin-1 (IL-1), IL-6, IL-8, tumor necrosis factor, and interferon-.gamma.. An interplay between these vasoconstrictors and vasodilators has been presumed to be the reason for the complex cardiovascular profile in endotoxin shock.
At present, diagnosis of septic shock involves monitoring the following clinical manifestations: (a) a body temperature greater than 38.degree. C. or less than 36.degree. C., (b) a heart rate greater than 90 beats per minute, and (c) a breath rate greater than 20 breaths per minute, and (d) an alteration in the white blood cell count greater than 12,000/mm.sup.3 or less than 4,000/mm.sup.3. The poor prognosis is, due at least in part to, the lack of reliable diagnostic tools to identify patients at greatest risk.
The relatively recent identification of nitric oxide as a signal transmitter in mediation of blood pressure regulation, neurotransmission, and macrophage-induced cytotoxicity and cytostasis has generated tremendous research interest. To understand the mechanisms by which nitric oxide, a diffusible free radical with a lifetime of a few seconds mediates various physiological and pathophysiological processes, it is necessary to have a method of detecting nitric oxide in real time near its sites of production and action. Several nitric oxide detection methods have been developed. These include chemiluminescence assay, oxyhemoglobin assay, GC-MS detection, and nitrosyl-hemoglobin formations detected by electron paramagnetic resonance (EPR) spectroscopy at liquid nitrogen temperatures. In addition, the production of nitric oxide can also be indirectly detected by measuring its end products NO.sub.2.sup.- /NO.sub.3.sup.-. However, none of these techniques in their present forms can be used for real time detection of nitric oxide in vivo or in the isolated body fluids.
As a result, there is a need for a method of detecting nitric oxide in real time in vivo or in isolated body fluids. In addition, because it is so often lethal, there also is a need for a fast and easy method of determining if a patient has septic shock.